The long-term objective of our research is to understand the mechanisms and physiological functions of Ca2+ signaling in mammalian cells. Ca2+ mobilization from intracellular stores represents an important cell signaling process. Of the three known Ca2+ mobilization second messengers, inositol 1,4,5-trisphosphate (IP3), cyclic ADP- ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP), NAADP is least characterized. Recent studies indicate that NAADP mobilizes Ca2+ from lysosome-related acidic organelles; however, the molecular identity of the Ca2+ release channels and the specific internal stores involved in NAADP-stimulated Ca2+ release remain elusive. The two pore channels (TPCs) belong to the voltage-gated ion channel superfamily. The three TPC genes encode proteins that are most closely related to the pore-forming subunit of voltage-gated Ca2+ and Na+ channels. Each TPC protein contains 12 putative transmembrane 1-helices with two potential pore loops. Our recent studies show expression of TPC1 and TPC3 on endosomal membranes and that of TPC2 on lysosomal membranes, suggesting that TPCs are most likely Ca2+- permeable channels of acidic organelles. We show that TPC2-enriched membranes bind to NAADP with a high affinity at low nanomolar concentrations and cells overexpressing TPC2 have enhanced response to intracellular application of NAADP. NAADP response is abolished by disrupting proton gradient of lysosomes and RNAi-mediated silencing of TPC2 expression, as well as genetic ablation of the Tpc2 gene in mice. Furthermore, the NAADP-elicited Ca2+ signal is coupled to Ca2+ release from the endoplasmic reticulum, suggesting a cross-talk between NAADP and IP3 receptors. The goals of the current project are to test the hypothesis that TPCs form NAADP receptors that mediate Ca2+ release from different endo-lysosome populations with in-depth characterization of these novel Ca2+ release channels (Aim 1), and to explore the functional cross-talk between NAADP-induced Ca2+ release from acidic stores and Ca2+ mobilization from endoplasmic reticulum, as well as additional Ca2+ signaling pathways (Aim 2). A multidisciplinary approach employing molecular biology, biochemistry, pharmacology, cell biology, and electrophysiology will be used to accomplish the two specific aims. These comprehensive studies should greatly enhance our understanding on NAADP signaling and shed new lights on the roles of this important cell signaling pathway in a broad spectrum of cell types and their involvement in normal human physiology and pathophysiology especially because lysosomal Ca2+ handling has been implicated in autophagy and lysosomal storage diseases. PUBLIC HEALTH RELEVANCE: Calcium ions are very important for cell signaling. Of the three second messengers known to induce Ca2+ release from intracellular Ca2+ storage pools, IP3, cADPR and NAADP, the mechanism and physiological function of NAADP-induced Ca2+ release are the least understood. The proposed project will show that two-pore channels are NAADP receptors expressed on endosomes and lysosomes and they play important functions in regulating Ca2+ release from acidic organelles and shaping Ca2+ signaling via cross-talking to Ca2+ release channels on endoplasmic reticulum.